Method for removing and/or detecting nucleic acids having mismatched nucleotides

Abstract

Provided herein, among other things, are various in vitro methods that involve cleaving dsDNA molecules that comprise a mismatched nucleotide using EndoMS. In some embodiments, the method may compriseligating a T-tailed double-stranded adapter to A-tailed double-stranded fragments of nucleic acid to produce ligation products that comprise adapter-ligated fragments and double-stranded adapter dimers that comprise a T:T mismatch at the ligation junction and cleaving both strands of the adapter dimers using EndoMS.

Description

Background technique [0001] In the process of next-generation DNA sequencing, DNA molecules to be sequenced are usually cut into fragments, repaired, and adapters of known sequences are ligated into an insert library. A critical step in sample preparation for DNA sequencing is the ligation of oligonucleotide adapters to a population of DNA fragments. DNA fragments are usually 3' tailed with dA to prevent self-ligation of library DNA. Adapters were designed with 3'-T overhangs to preferentially ligate to the 3'-dA of the fragments. During ligation, adapters are in excess relative to fragments to maximize ligation efficiency. Typically, a molar ratio of 10:1 adapter:insert is recommended to maximize ligation efficiency (Head, et al., Biotechniques, 2014, 56, 61-68). However, the use of higher adapter:fragment ratios leads to "adaptor dimers" due to the fact that the adapters are directly self-ligated to each other rather than to the library insert. [0002] The problem of ad...

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Problems solved by technology

However, the main source of adapter dimers - adapter dimers containing T-T mismatches - is not described in this publication

Furthermore, not only can the guide RNA potentially hybridize to the genomic sequence to produce undesired off-target cleavage, but the introduction of the guide RNA molecule into amplification and / or sequencing reactions can potentially cause other artifacts (artefacts)

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